Rapid, high-throughput compound screening assays have revolutionized the field of drug discovery. Automated drug discovery assays measure changes in a variety of biochemical pathways in vitro. For example, microarray assays simultaneously assess the effect of thousands of compounds on a particular biochemical pathway in vitro. However, such automated drug screening assays are not readily available for assessing the in vivo effects of multiple compounds on complex multicellular organisms. In particular, assessing the effect of one or more compounds on the development or physiology of a multicellular organism remains a tedious manual task requiring hundreds of hours of labor by highly skilled technicians.
Intact multicellular organisms, such as the nematode Caenorhabditis elegans, the fruit fly Drosophila melanogaster, or the zebrafish Danio rerio are frequently used as model systems to help understand the function of human genes that have been implicated in disease. Human gene homologs are often identified in these model organisms and provide valuable tools for studying the biological function of the genes in vivo. Such mutations frequently result in an easily observable phenotypic change in the model organism. Furthermore, it has been shown that certain pharmacological compounds collaterally produce optically detectable changes in these mutant organisms. These changes can be used to identify specific compounds that interact with a particular gene product in vivo. The addition of such functional genomic techniques to the repertoire of molecular biology and biochemistry methods can greatly accelerate the drug discovery process.
Mutants of intact organisms are used as a new class of methods for in vivo drug screening of libraries of potential pharmacological compound produced through the use of combinatorial chemical methods. With these organisms, one can identify targets for drug intervention without the need to completely understand the complex biochemical pathways that relate the genome to the phenotype. In addition, investigators can annotate drug libraries for toxicity, non-specific activity, or cell membrane permeability by observing their behavior in intact organisms. In this way, toxic or ineffective libraries and/or library members can be discarded at an early stage of testing without wasting valuable resources. This allows rapid and economical screenings of the compound libraries for new and useful therapeutic compounds, while limiting politically controversial testing on mammals.
While model organisms such as C. elegans, D. melanogaster, and D. rerio have been proven useful in the study of human disease, they have not yet been successfully used in the field of high speed, high throughput drug discovery. This presents a significant hindrance to investigators that need to search through thousands of multicellular organisms for the phenotype of a new mutation or for a response to a panel of sample drugs. For example, with today's molecular biology techniques, a large laboratory can produce deletion mutations in a multicellular test organism at a rate of 20 to 30 per month. In order to evaluate the effect of each member of a chemical compound library (that frequently contains 100,000 discrete compounds) on a single deletion mutant, one must manually manipulate and deposit a precise number of organisms of the mutant strain at the same developmental stage into various containers, such as wells of a microtiter plate array. Wild-type or deviants from the desired mutant strain, or organisms at a different development stage must be eliminated. The use of such slow, manual methods for the selection and deposition of organisms of the proper type greatly delays the entire process of drug discovery. Moreover, manual methods rely on pipettes that dispense accurate volumes of fluid but not accurate numbers of organisms. In many studies, where reproduction rate is altered by the mutation, it is necessary to begin with an exact and known number of multicellular organisms in each well. This is, at best, a daunting requirement.
The effect of a therapeutic compound or toxic environment on the mutant strain or expression system can be determined by identifying changes in the spatial pattern of fluorescence or staining. Fluorescent protein genes are typically used as reporters for gene expression in a wide variety of organisms (Tsien, R. Nature Biotechnology (1999) 17: 956–57). For example, green fluorescent protein (GFP) is used as a reporter gene to indicate that an inserted gene has been expressed. The expression of the fluorescent protein usually occurs in a specific spatial pattern within a multicellular organism. Discrimination of one pattern from another is currently carried out manually using a fluorescent microscope. Like the selection and deposition step, this is an extremely tedious task requiring a significant number of workers that are trained at high academic levels.
Prior art methods of selecting multicellular organisms have relied on instruments that performed a “slit-scan” of whole organisms as they passed through the analysis zone of a laser. Methods of detecting fine detail in slit-scanning have relied on apparatuses that utilize diffraction limited optics to create narrow line focus and image plane masks to act as optical spatial filters. This narrow line of focus is sufficient for analyzing single cells, but is insufficient for detecting and spatially locating a particular feature against the more complex background profile of light scatter and autofluorescence presented by a multicellular organism. For example, the diameter of a mature C. elegans is approximately 70 micrometers. This means that the background autofluorescence from a nematode is approximately ten times that from a white blood cell (about seven micrometers in diameter), while a fluorescence reporter signal from a single C. elegans cell is no greater than that from a single blood cell. In the case of D. melanogaster (fruit fly) larvae, the situation is even worse because the diameter of an advanced stage larva is of the order of one millimeter, which means that autofluorescence is much more than a hundred times greater than in single blood cells. This means that experimentally created, fluorescent features along the axis of a multicellular organism may produce a much weaker optical signal than the autofluorescence background. One can imagine that an axial profile of auto fluorescence with very high peaks and valleys would effectively mask an experimentally created fluorescence feature.
Flow instruments have been used to count the number of nematodes in a fluid volume. Such a device was described by Byerly et al (Byerly,et al., Rev. Sci. Instrum. (1975) May 46(5): 517-22), where a flow cytometer employed sheath flow to orient nematodes along the direction of flow so that their size could be measured and organism-by-organism counts could be made by an electrical impedance method. The device was similar to a commercial Coulter counter. The use of the impedance sensor, which can only estimate overall size, and cannot spatially resolve localized features along the major axis of the organism limits the Byerly et al. instrument. In addition to this limitation, the Byerly et al. instrument could not select and deposit (sort) specific organisms.
U.S. Pat. Ser. No. 6,400,453 issued Jun. 6, 2002, which is incorporated herein by reference, describes an instrumentation system for the rapid analysis and sorting of multicellular organisms using optical characteristics such as light scatter and fluorescence to classify each organism in a flowing stream. A single value of fluorescence intensity at a given emission wavelength is detected and assigned to each organism. However, this instrument reports only the intensity, not the position of fluorescence along the major (long) axis of the organisms.
An optical flow instrument for analyzing elongate organisms such as plankton with widths of 500 μm and lengths over 1000 μm has also been described with sheath flow to achieve orientation of the plankton. (J. C. Peters, G. B. Dubelaar, J. Ringelberg, and J. W. Visser, “Optical Plankton Analyser: a Flow Cytometer for Plankton Analysis, I: Design Considerations” Cytometry Sep. 10, 1989 (5): 522–528; and G. B. Dubelaar, A. C. Groenwegen, W. Stokdijk, G. J. van den Engh, and J. W. Visser, “Optical Plankton Analyser: a Flow Cytometer for Plankton Analysis, II: Specifications”, Cytometry Sep. 10, 1989 (5): 529–539). The size range of the plankton used in these optical flow cytometers is similar to that encountered with C. elegans nematodes, fruit fly larvae, and zebrafish embryos; however, there is no provision that avoids the ambiguous light scatter signals that are eliminated by the present invention.
There exists the need for a high-speed system for automatically identifying and physically selecting multicellular organisms with certain spatially distinct, optically detectable, phenotypic characteristics from mixed populations. Such a system must have the ability to locate and measure the intensity and position of experimentally created optical features in the presence of overwhelming autofluorescence.